The present invention relates to a method of treating metal surfaces to enhance the bio-compatibility and/or physical characteristics of said surfaces. The invention also relates to bio-compatible metal articles. The invention is particularly relevant to surfaces of medical devices.
Many medical techniques are known in which human or animal blood is brought into contact with foreign surfaces, either within the body or outside the body. In some situations, usually due to mechanical characteristics, it is necessary to use metallic surfaces, as required by coronary stents (vascular endoprostheses) located within arteries or, for example, within heat exchanger assemblies external to the body. Thus, in the first application, the mechanical strength of the metal object is required whereas in the external application it is the heat transfer characteristics that are required. However, in both applications, blood or related blood products are brought into contact with metal surfaces, which may in turn have detrimental effects upon the blood itself.
The Problem of Clotting
When presented to foreign surfaces, blood has a tendency to clot. It is known that blood activation in response to contact with a foreign surface occurs by the intrinsic pathway (see FIG. 2 of Johan Riesenfeld et al, Surface Modification with Functionally Active Heparin, Medical Device Technology, March 1995 pages 24-31, the disclosure of which is incorporated herein by reference), triggered by the conversion of Hageman (F)XII to an active enzyme, FXIIa. This then initiates the sequential activation of coagulation factors FXI, FIX and FX and finally FXa converts prothrombin into enzymatically active thrombin, which precipitates the soluble plasma protein fibrinogen into a solid fibrin clot. The coagulation system is under the control of a series of regulatory mechanisms in the blood and the vascular wall, the most important being the plasma coagulation inhibitor, antithrombin (III).
Heparin is a naturally occurring substance that consists of a polysaccharide with a heterogeneous structure and a molecular weight ranging from approximately 6000 to 30000 Dalton (atomic mass units). It prevents uncontrolled clotting by suppressing the activity of the coagulation system through complexing with antithrombin (III), whose activity it powerfully enhances. Approximately one in three heparin molecules contains a sequence of highly specific structures to which antithrombin binds with high affinity. When bound to the specific sequence, the coagulation enzymes are inhibited at a rate that is several order of magnitude higher than in the absence of Heparin. Thus, the heparin molecule is not in itself an inhibitor but acts as a catalyst for natural control mechanisms without being consumed during the anticoagulation process. The catalytic nature of heparin is a desirable property for the creation of a bio-active surface, because the immobilised heparin is not functionally exhausted during exposure to blood but remains a stable active catalyst on the surface.
A method for making nonthrombogenic surfaces is disclosed in U.S. Pat. No. 3,634,123. A method for reducing thrombosis of blood, induced by contact with foreign surfaces, is shown in which the surface are treated with a cationic surface active agent and a conventional anticoagulant such as heparin. The technique disclosed in this patent is appropriate for plastic surfaces but cannot be extended to metal surfaces.
The Benestent II Group at the Department of Cardiology, University Hospital Rotterdam have developed a heparin coated Palmaz-Schatz stent, in which an end point of the heparin molecule is covalently coupled to an underlying polymer matrix, similar to the type manufactured under the trade mark Carmeda Bioactive Surface by CBAS Carmeda Inc, Sweden. The process consists of four stages:
etch the metal surface
introduce a poly-amino layer which is ionically attached to the surface
covalently bond the functional amino groups to the aldehyde groups of partially degraded heparin molecules
chemically stabilise the bonded heparin by use of a reducing agent.
An advantage of this known approach is that it allows heparin molecules to be attached to the poly-amino layer in a relatively friendly chemical environment. However, the poly-amino layer is only physically attached to the conditioned metal surface and as such the strength of the attachment is somewhat dubious. Thus, in continuous use within the body, there is a risk of heparin or similar molecules becoming detached thereby reducing the effectiveness of the stent, which in turn may require further surgery. Similarly, in external applications, the effectiveness of the device may degrade and this degradation may be accelerated if the device has to be cleaned under particularly harsh conditions. Finally anti-coagulant coating methods generally incur relatively high manufacturing costs.
U.S. Pat. No. 5,356,433 discloses the treatment of a stent or other medical device by the alleged formation of covalent linkages between a biologically active agent and a metallic surface. In one example tantalum stents were primed with a solution in ethanol of N-(2-aminoethyl-3-aminopropyltrimethoxysilane so that a bond was formed between the tantalum oxide layer on the surface of the stents and the silicon of the silane on curing at 110xc2x0 C. Heparin is then coupled to the amino groups using 1,3-ethyldimethyl-aminopropyl carbodiimide (EDC). In a second example, an ethanolic solution of an amiofunctional polymeric silane, trimethylsilylpropyl substituted polyethyleneimine is bonded to the surface of tantalum stents, also with curing at 110xc2x0 C., after which heparin was coupled to the coating using EDC. Other examples use stainless steel wire, platinum tungsten wire and aminopropyl-trimethoxysilane as primer. However, priming has to be carried out with heating. The present applicants consider that covalent bonds to the metal surface are not formed under the conditions described. The reason is that the water which is inevitably present in the ethanol hydrolyses the linkages between the methoxy groups and silicon and because the reaction between the trimethoxysilane groups and surface oxide requires a catalyst which is absent. Furthermore, the heparin is coupled to the priming layer directly and not by polymeric or oligomeric spacer arms, is not sterically available, and will therefore not exhibit its full anti-coagulant activity. U.S. Pat. No. 5,607,475 reports that the use of aminosilanes in coatings on metal or glass surfaces has not been good at producing a surface with a high level of both bio-effectiveness and stability.
U.S. Pat. No. 5,607,475 discloses an endoprosthesis having a metal surface for contact with body fluids, the metal surface having a coating thereon comprising:
(a) a silane which includes a vinyl functionality, the silane being adherent to the metal surface so that the vinyl functionality is pendant from the surface;
(b) a graft polymer, the graft polymer being covalently bonded with the pendant vinyl functionality of the adherent silane, the graft polymer being simultaneously formed and bonded to the pendant vinyl functionality by free radical reaction initiated by an oxidising metal with at least one ethylenically unsaturated monomer selected from the group consisting of acrylamide and acrylic acid;
(c) a polyamine spacer covalently attached to the graft polymer; and
(d) a biomolecule covalently attached to the spacer.
The preferred primer is trichlorovinylsilane which is applied in xylene. However, under these conditions, the primer is merely physically held to the metal surface and does not form a chemical bond with oxide on the metallic surface. The procedure for subsequent attachment of heparin is lengthy and complex. The method described is neither effective nor practical, and the information and belief of the present applicants is that it has not been put into practice.
WO 97/07834 acknowledges that in order to obtain truly anti-thrombogenic surfaces, proper immobilisation of the biomolecules is the key and that the binding of a base or primer layer to metal or glass surfaces presents a problem because of the difficulty of forming covalent bonds to the surface. The priming step uses trichlorovinylsilane in xylene and therefore does not form a covalent attachment with the metal surface/ Subsequent attachment of heparin is by an elaborate multi-step procedure.
The Problem of Restenosis
Vascular restenosis is a serious complication of percutaneous transluminal coronary angioplasty (PCTA). Amongst patients who undergo this procedure, re-occlusion within 3-6 months can occur in about 35% of cases. Damage to the lining of an artery can give rise to uncontrolled proliferation of smooth cells, which gives rise to the restenosis. Intensive research has been carried out to find drugs that can avoid restenosis by inhibiting smooth cell proliferation, and stents have also been installed in an attempt to reduce the rate of restenosis.
Stents were introduced into clinical practice in 1986 in order to treat abrupt or threatened vessel closure and to prevent restenosis after angioplasty. However, when a stent is installed during angioplasty, in-stent restenosis can occur. The reasons why the arteries of some patients react to form a restenosis whereas those of others do not are not understood. Typically a patient may develop chest pain about two months after surgery, and an angiogram will reveal a blockage in the stent. Current procedures to alleviate this problem involve using a rotational device to clean out the stent, installation of a second stent within the first, and subjecting the in stent restenosis to radiotherapy. If these further procedures should fail, the patient may need to undergo by-pass surgery.
One problem with which the invention is concerned is how to provide a primer layer on a metal, glass or ceramics surface which is sufficiently durably attached that it can withstand prolonged contact with blood or other biological fluids, that enables bio-compatible hydrophilic chains or spacer arms to be grafted onto the primer layer, and that allows easy and effective attachment of heparin or other biologically active molecules.
In one aspect, the present invention provides a method of treating a metal, glass or ceramics article having at its surface oxide or hydroxide to enhance the bio-compatibility and/or physical characteristics of the surface, said method comprising the steps of:
priming said surface by means of functional molecules each of has at least one alkoxysilane group which can form at least one first covalent bond by reaction with the oxide or hydroxide of said surface and at least one other group which can participate in free-radical polymerisation, the priming being carried out by contacting said surface in an aprotic organic solvent with said functional molecules and with an acid which facilitates formation of said first covalent bond; and
forming chains covalently attached to said other group of the functional molecules by free-radical graft polymerisation of at least one polymerizable monomer which imparts hydrophilic properties to said chains. The graft polymerisation is preferably a free radical polymerisation because of ease of production on a commercial scale and because of speed of the reaction. However, it could be an addition polymerisation which is ionically initiated.
In the presence of an acid, priming can be carried out under mild conditions, and thereafter formation of spacer arms and attachment of heparin or other biomolecules (if required) can be carried out under mild aqueous conditions. Attachment of heparin or other biological macromolecules may be carried out simultaneously with formation of the spacer arms, or the spacer arms may be provided with attachment sites for heparin or other biological molecules as they are formed, after which the heparin or other biological macromolecules are attached in a separate operation.
According to a second aspect of the present invention, there is provided a bio-compatible metal, glass or ceramics article a surface of which is primed with residues derived from functional molecules covalently bonded to said surface, wherein said functional molecule residues are polymers having more than one alkoxysilane group per molecule and wherein said surface carries bio-compatible hydrophilic polymer chains covalently bonded to said functional molecule residues.
A further problem with which the invention is concerned is the provision of a stent which when implanted in the human or animal body gives rise to a reduced incidence of restenosis.
That problem is solved, according to a further aspect of the invention by the provision of a stent which has a polymer coating covalently bonded to its surface, a molecular species which imparts thrombosis resistance covalently bonded in or to said coating, and a molecular species which inhibits restenosis held by ionic attraction in or to said coating.
An alternative solution to the problem provides a stent which has a polymer coating covalently bonded to its surface, wherein the coating has radio-labelling in an amount effective to inhibit restenosis.
A further solution to the above mentioned problem is to provide a stent which has a polymer coating covalently bonded to its surface, wherein the coating is radio-labelled and wherein a molecular species which inhibits restenosis is held by ionic attraction on ot to said surface, wherein the amounts of radio-labelling and of the molecular species are sufficient to inhibit restenosis.
Priming the Surface
Where they are monomeric, said functional molecules may be of any of the formulae:
CH2xe2x95x90CHR1xe2x80x94(CH2)nxe2x80x94Si(OR2)3 
CH2xe2x95x90CHR1xe2x80x94(CH2)nxe2x80x94Si(OR2)2R3 or
CH2xe2x95x90CHR1xe2x80x94(CH2)nxe2x80x94Si(OR2)R3R4 
wherein R1 represents a hydrogen atom or an alkyl group, R2, R3 and R4 represent an alkyl group and n is 0 or is a positive integer. In the above molecules, preferably R1 represents hydrogen, methyl or ethyl and R2, R3 and R4 represent methyl or ethyl and the value of n is from 0 to 6. Other values of R3 and R4 e.g. hydroxyl or chloride are possible provided that the bond formation is not interfered with.
Preferably said vinylfunctional silane molecules are oligomers or polymers, and preferably said vinylfunctional silane molecules become bonded to said surface at a plurality of locations.
In a preferred group of oligomers or polymers, the functional molecules comprise a [xe2x80x94Sixe2x80x94Oxe2x80x94]n chain having alkoxy groups directly attached to the silicon atoms and having olefinically unsaturated groups attached directly or via linking groups to the silicon atoms. Preferably the functional molecules have vinyl and alkoxy groups attached to the silicon atoms of the chain.
The molecules of a further preferred group of functional molecules have:
an oligomeric or polymeric chain based on carbon atoms, which chain may also include nitrogen or oxygen atoms;
one or more alkoxysilane or alkylalkoxysilane groups attached to the chain for forming covalent bonds with oxide or hydroxide of the surface; and
one or more olefinically unsaturated groups which can participate in free radical polymerisation. The above functional molecules preferably have trialkoxysilane or dialkoxyalkylsilane groups, alkyl preferably being methyl or ethyl.
Particular functional molecules which may be used include one or more of:
3-(trimethoxysilyl) propyl methacrylate;
vinylmethoxysiloxane oligomer;
diethoxymethylsilyl-modified polybutadiene
triethoxysilyl-modified polybutadiene.
Formation of Spacer Arms by Graft Polymerisation
The graft polymerisation reaction to form hydrophilic spacer arms preferably involves free-radical polymerisation including polymerising a plurality of types of polymerisable molecules to form polymer chains of said spacer arms. The polymer chains may include molecular units derived from acrylamide, and may provide sites for covalent or ionic bonding to a bio-active molecule. Such bonding sites may be provided by amino groups e.g. using as monomer or co-monomer 3-aminopropyl methacrylamide. Additionally or alternatively, the bonding sites may be carboxyl groups. A polymerizable monomer that provides sites of both type is dimethyl(methacryloyloxyethyl)(3-sulfopropyl)ammoniumbetaine (SPE) which is of formula:
CH2xe2x95x90CHCH3xe2x80x94COxe2x80x94Oxe2x80x94CH2 CH2xe2x80x94+N(CH3)2xe2x80x94CH2 CH2 CH2xe2x80x94SO331 
In a further preferred embodiment, at least one type of said polymerisable molecules is suitable for additionally bonding to a bio-active molecule. In an alternative embodiment at least one of said polymerisable molecules is a modified bio-active molecule e.g. a molecule with anti-thrombolytic properties. In a further preferred embodiment, said bio-active molecule is heparin, or in the case of a modified bio-active molecule, a heparin derived molecule.
Stents and Restenosis
In a further preferred embodiment, said metal surface is a surface of a medical device, e.g. a stent. In addition to having an anti-thrombogenic compound, the stent preferably has attached thereto a compound that is effective to inhibit smooth muscle cell proliferation and restenosis. Such compounds can be covalently attached to the coating, but are preferably held by ionic bonds so that they become gradually released and are available for absorption by cells. Any physiologically acceptable at least slightly water-soluble compound that is active against smooth muscle cell proliferation and restenosis and that can form an ionic bond with the spacer arms of the covalently bonded stent coating could in principle be used. Although high molecular weight materials may be used for this purpose e.g. an anti-sense DNA fragment, simple low molecular weight compounds will be more usually employed. Amongst the classes of compound which have been reported to have appropriate properties there may be mentioned the following:
(a) Anthraquinones, e.g. the symmetrical 1,4-bis(substituted-amino)-5,8-dihydroxyanthroquinones of U.S. Pat. No. 4,197,249 and pharmaceutically acceptable salts thereof, of which mitoxantrone and its salts is preferred. See also daunorubicin, doxorubicin, and related compounds and their pharmaceutically acceptable salts.
(b) Imidazoles, e.g. chlotrimazole, miconazole, econazole and their pharmaceutically acceptable salts whose ability to inhibit proliferation of smooth muscle cells is disclosed in U.S. Pat. Nos. 5,358,959, 5,591,763 and 5,643,936.
(c) Substituted benzimidazoles and their pharmaceutically acceptable salts, e.g. as disclosed in U.S. Pat. No. 5,763,473 and EP-A-0882718.
(d) Raloxifene-type benzothiophene compounds and salts thereof, e.g as disclosed in U.S. Pat. Nos. 5,462,937, 5,457,113, 5,643,876, 5,688,796 and 5,760,030.
(e) Carbazole derivatives and their pharmaceutically acceptable salts, e.g. as disclosed in U.S. Pat. No. 5,643,939.
(f) Naphthyl compounds and their pharmaceutically acceptable salts, e.g. as disclosed in U.S. Pat. Nos. 5,484,796 and 5,691,353.
(g) Retinoids containing salt-forming groups, and pharmaceutically acceptable salts thereof, e.g. as disclosed in U.S. Pat. No. 5,798,372.
(h) Thiazoles and their pharmaceutically acceptable salts, eg as disclosed in EP-A-0928793
(i) Phenylcyclohexylcarboxamides and their pharmacologically acceptable salts, e.g. as described in U.S. Pat. No. 593,598
(j) Tranilast and its pharmaceutically acceptable salts, e.g. as described in WO 98/29104.
A further possibility for inhibiting restenosis is to incorporate radio-labelling into the stent coating e.g. by incorporating into the coating heparin labelled with 35S. A yet further possibility is both incorporate into a stent coating both a radio-labelled compoud (e.g. a compound labelled with with 35S) and a molecular species which inhibits restenosis.